The proposed work will evaluate an innovative technology for profiling cancer initiator cells (also known as cancer stem cells) to identify markers that can be used in diagnostics and to identify molecular targets that can guide drug development programs. Oral squamous cell carcinoma (OSCC), which is diagnosed in > 25,000 patients in the US each year, has been steadily increasing over the past 5 years. OSCC causes significant morbidity as a result of speech and swallowing difficulties following surgical resectio and radiation treatment. Moreover, recurrent and metastatic OSCC is poorly responsive to cytotoxic chemotherapy. Hence, there remains a pressing need for strategies that aid earlier detection and for the development of new drug therapies that have higher treatment efficacy. The development and progression of oral cancers (among others) relies on a small population of cancer stem cells which can not only regenerate the tumor bulk following local treatment but also contribute to cancer recurrence by being particularly refractory to standard chemotherapy. The proposed work will apply a novel peptide array technology to profile lysates of oral epithelial cells, OSCC cells and OSCC cells that have undergone EMT to identify patterns of deacetylase activity that may underlie the CSC phenotype. The technology is based on the SAMDI mass spectrometry technique that uses matrix- assisted laser desorption-ionization mass spectrometry to analyze an array of peptides immobilized to self- assembled monolayers. SAMDI mass spectrometry detects the mass of the peptide-alkanethiolate conjugate and therefore can identify changes in acetylation of the peptides. The proposed work will apply this method to profile deacetylase activities in cell cultures and identify differences in the activity profiles between oral keratinocytes, OSCC cells, OSCC cells that have undergone EMT, and in CSCs. The first Aim will develop peptide arrays for profiling acetylation activities in cell lysate and will validate the reproducibility of the experiment for the lysate and will use selective inhibitors to parse the activities to understand which specific enzymes contribute to the activity profiles. The second aim will apply the arrays to compare the acetylation activities in OSCC cells to those present in the CSC population. The goal of this R21 proposal is to validate the use of the SAMDI technology for identifying molecular activities that underlie the generation and maintenance of cancer stem cells. The work has the broader goal of introducing an innovative strategy for understanding the regulation of cancer stem cells and may lead to new diagnostic approaches and targets for drug development.